The present invention relates generally to a method and system for measuring the activity of enzyme bound to a solid surface, and more particularly to a spectrophotometric method and system for absorbance measurements in two-phase enzyme immunoassays.
Many immunoassay methods currently in favor in the clinical laboratory require, as their last step, spectrophotometric measurement of the activity of an enzyme covalently linked as a label to one of the reactants in the antigen-antibody reaction. In one form of such immunoassay, an antibody specific for the analyte is immobilized or fixed on the surface of a solid phase, such as a polymeric bead or the wall of a test tube. When incubated with the analyte, the antibody fixes the analyte through an antigen-antibody reaction. A second antibody, labeled by covalent linkage with an enzyme and also directed against the analyte, is added in solution to the incubation mixture. Through its reaction with the analyte, it too becomes bound to the surface. The quantity of enzyme bound is then directly proportional to the quantity of analyte. This enzyme is generally measured by its activity: the rate of the reaction is catalyzes when incubated with a predetermined concentration of its substrate under prescribed conditions. This rate, in turn, is measured by measuring the change in concentration with time of either the product of the reaction or the precursor reactant. Although many methods can be used to measure the activity of an enzyme in solution, procedures for measuring the enzyme bound to a surface are comparatively more difficult and subject to interference by the surface and by the need to supply more than the usual stirring or mixing of the reaction mixture. It is toward improving the measurement of this enzyme activity that the invention described here is directed.
An example of an assay employing this approach is the "TANDEM-E CKMB" immunoenzymetric assay (available from Hybritech Inc., San Diego, Calif. 92121), which is used to measure the serum concentration of one of the isoenzymes of creatine kinase (CK). CK, an enzyme that catalyzes the reversible phosphorylation of creatine by adenosine triphosphate, appears in serum of patients in higher than usual concentrations in the hours and days following acute myocardial infarctions. Its measurement is used in the diagnosis and monitoring of patients with this disease. The different isoenzymes of CK are composed of the two enzymatically active subunits, termed M and B, in the three possible dimeric combinations: MM, MB and BB. The different isoenzymes are separable by electrophoresis and chromatography and may also be distinguished by reaction with specific antibodies. They are found in different concentrations and ratios in various tissues of the body: the MM chiefly in skeletal and cardiac muscle, the MB chiefly in cardiac muscle, and the BB chiefly in brain and tissues of endodermal origin. In blood serum, CK is in higher than usual concentration in diseases involving ischemia or destruction of tissues in which the enzyme is in high concentration. In the hours and days after the onset of ischemia and destruction of cardiac muscle that is characteristic of acute myocardial infarction, manyfold increases in concentration of total CK, CK-MM, and CK-MB are characteristically measurable in serum. Since similar elevations of serum CK-MM, the major component of total CK activity, are noted in many other conditions that affect skeletal muscle, and these are much more frequent, elevation of CK-MB isoenzyme is much more specific for myocardial infarction and for that reason more diagnostically useful.
In the Tandem assay procedure, serum is reacted with a plastic bead (the solid phase member) coated with a monoclonal antibody directed toward a unique antigenic site on the M subunit of the CK MB molecule, and with a second monoclonal antibody, labeled with an enzyme such as alkaline phosphatase, that is directed toward a different antigenic site on the B subunit of the same CK-MB molecule. In the presence of CK-MB, both antibodies react, sandwiching the CK MB between them, and the enzyme label of the second antibody becomes immobilzed on the surface of the bead.
The bead is then washed to remove unbound labeled antibody and the remaining bound enzyme label is measured by incubating the bead in a solution of its substrate for a period of some 30 minutes, at a controlled temperature (e.g. 37.degree. C.). The reaction is then stopped and the quantity of substrate reacted is determined colorimetrically. With p-nitrophenyl phosphate, a commonly used substrate for the alkaline phosphatase labeling enzyme, the quantity of p-nitrophenol released is measured as the absorbance of the solution at 405nm, which is determined primarily by the concentration of p-nitrophenol. Through the sequence of antigen-antibody reactions described above, this absorbance is directly proportional to the concentration of CK-MB in the test sample.
The enzymatic reaction occurs at the surface where the labeling enzyme is bound. Unless the solution is stirred continuously, which is generally inconvenient, the reaction product tends to remain in high concentration near the surface and is not uniformly distributed throughout the incubation mixture. To measure the quantity of product produced, the "TANDEM-E CK MB" procedure, and indeed all of the other currently available two phase, enzyme linked immunoassay procedures, measure the absorbance only once, after the enzymatic reaction catalyzed by the labeling enzyme has progressed for a predetermined period of time. The reaction is then stopped, generally by dilution with reagents which change the pH of the mixture or remove a necessary cofactor such as magnesium ion by chelation. Once the reaction has been terminated, the solution is stirred to ensure that the measured product is uniformly distributed throughout the solution and the absorbance of the supernatant solution, away from the bead, is measured.
While offering some advantages, end point assays of enzyme activity present several important difficulties. First, the result cannot be read until the pre set incubation period is termined, some 30 minutes after the start of the enzymatic reaction in the procedure described above. Second, when the concentration of reaction product is low, corresponding to a low concentration of analyte in the sample, interference caused by unavoidable background absorbance of the sample in the solution can become relatively significant; detection of small quantities of analyte then becomes of doubtful validity. This is the reason the reaction is generally allowed to proceed long enough to ensure production of substantial quantities of reaction product so that its concentration becomes high compared to those of interfering compounds. When the concentration of analyte is small, even this measure may not be completely effective. Third, assays made on the basis of single spectrophotometric measurements are inherently imprecise. Fourth, although conditions are chosen that favor constancy of the reaction rate upon which the enzyme activity is based, deviations from constancy cannot be detected when the assay is based on measurement at only one time.
Accordingly, it is an object of the present invention to provide a spectrophotometric method for kinetic absorbance measurements in two phase enzyme immunoassays.
Another object of the present invention is to provide such a method which allows the assays to be performed in less time than required for conventional end-point assays, with greater sensitivity, enhanced precision, and confirmation of constancy of reaction rate.
A further object of the present invention is to provide such a method which can be practiced on existing spectrophotometric systems with only minor physical modification.
Yet another object is to provide apparatus for performing such a method.